Cryopreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification.
Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.
Bhakta
Gajadhar
G
Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical Drive, Singapore 117597, Singapore.
Lee
Kong Heng
KH
Magalhães
Raquel
R
Wen
Feng
F
Gouk
Sok Siam
SS
Hutmacher
Dietmar W
DW
Kuleshova
Lilia L
LL
D000464:Alginates / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D008863:Microspheres; D008903:Minerals; D010012:Osteogenesis; D013194:Staining and Labeling; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D034421:Sus scrofa
D000464:Alginates; D006603:Hexuronic Acids; D008903:Minerals; D020723:Glucuronic Acid; D005337:Fibrin; D002118:Calcium